Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the <i>FAD2</i> and <i>FATB</i> Multigene in Soybean

CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. I...

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Autores principales:	Won-Nyeong Kim, Hye-Jeong Kim, Young-Soo Chung, Hyun-Uk Kim
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	CRISPR/Cas9 multiple sgRNA polycistronic-tRNA-gRNA Golden Gate cloning type IIS restriction enzyme <i>FAD2</i> Botany QK1-989
Acceso en línea:	https://doaj.org/article/3532a30c59e14ccd946ade08fa5b99ad
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spelling	oai:doaj.org-article:3532a30c59e14ccd946ade08fa5b99ad2021-11-25T18:47:31ZConstruction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the <i>FAD2</i> and <i>FATB</i> Multigene in Soybean10.3390/plants101125422223-7747https://doaj.org/article/3532a30c59e14ccd946ade08fa5b99ad2021-11-01T00:00:00Zhttps://www.mdpi.com/2223-7747/10/11/2542https://doaj.org/toc/2223-7747CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of <i>FAD2</i> and <i>FATB</i> in soybean using this protocol. These three vectors were transformed into soybeans using the <i>Agrobacterium-</i>mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the <i>FAD2</i> and <i>FATB</i> multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants.Won-Nyeong KimHye-Jeong KimYoung-Soo ChungHyun-Uk KimMDPI AGarticleCRISPR/Cas9multiple sgRNApolycistronic-tRNA-gRNAGolden Gate cloningtype IIS restriction enzyme<i>FAD2</i>BotanyQK1-989ENPlants, Vol 10, Iss 2542, p 2542 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR/Cas9 multiple sgRNA polycistronic-tRNA-gRNA Golden Gate cloning type IIS restriction enzyme <i>FAD2</i> Botany QK1-989
spellingShingle	CRISPR/Cas9 multiple sgRNA polycistronic-tRNA-gRNA Golden Gate cloning type IIS restriction enzyme <i>FAD2</i> Botany QK1-989 Won-Nyeong Kim Hye-Jeong Kim Young-Soo Chung Hyun-Uk Kim Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the <i>FAD2</i> and <i>FATB</i> Multigene in Soybean
description	CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of <i>FAD2</i> and <i>FATB</i> in soybean using this protocol. These three vectors were transformed into soybeans using the <i>Agrobacterium-</i>mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the <i>FAD2</i> and <i>FATB</i> multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants.
format	article
author	Won-Nyeong Kim Hye-Jeong Kim Young-Soo Chung Hyun-Uk Kim
author_facet	Won-Nyeong Kim Hye-Jeong Kim Young-Soo Chung Hyun-Uk Kim
author_sort	Won-Nyeong Kim
title	Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the <i>FAD2</i> and <i>FATB</i> Multigene in Soybean
title_short	Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the <i>FAD2</i> and <i>FATB</i> Multigene in Soybean
title_full	Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the <i>FAD2</i> and <i>FATB</i> Multigene in Soybean
title_fullStr	Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the <i>FAD2</i> and <i>FATB</i> Multigene in Soybean
title_full_unstemmed	Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the <i>FAD2</i> and <i>FATB</i> Multigene in Soybean
title_sort	construction of multiple guide rnas in crispr/cas9 vector using stepwise or simultaneous golden gate cloning: case study for targeting the <i>fad2</i> and <i>fatb</i> multigene in soybean
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/3532a30c59e14ccd946ade08fa5b99ad
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Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the <i>FAD2</i> and <i>FATB</i> Multigene in Soybean

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