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Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research
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 reporte...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570347/ https://www.ncbi.nlm.nih.gov/pubmed/32854250 http://dx.doi.org/10.3390/plants9091090 |
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author | Gong, Qi Wang, Bin Lu, Xubiao Tan, Jiantao Hou, Yuke Liu, Taoli Liu, Yao-Guang Zhu, Qinlong |
author_facet | Gong, Qi Wang, Bin Lu, Xubiao Tan, Jiantao Hou, Yuke Liu, Taoli Liu, Yao-Guang Zhu, Qinlong |
author_sort | Gong, Qi |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7570347 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-75703472020-10-28 Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research Gong, Qi Wang, Bin Lu, Xubiao Tan, Jiantao Hou, Yuke Liu, Taoli Liu, Yao-Guang Zhu, Qinlong Plants (Basel) Article 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. MDPI 2020-08-25 /pmc/articles/PMC7570347/ /pubmed/32854250 http://dx.doi.org/10.3390/plants9091090 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Gong, Qi Wang, Bin Lu, Xubiao Tan, Jiantao Hou, Yuke Liu, Taoli Liu, Yao-Guang Zhu, Qinlong Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research |
title | Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research |
title_full | Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research |
title_fullStr | Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research |
title_full_unstemmed | Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research |
title_short | Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research |
title_sort | nicking endonuclease-mediated vector construction strategies for plant gene functional research |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570347/ https://www.ncbi.nlm.nih.gov/pubmed/32854250 http://dx.doi.org/10.3390/plants9091090 |
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