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CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean

At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for...

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Autores principales: Cai, Yupeng, Chen, Li, Sun, Shi, Wu, Cunxiang, Yao, Weiwei, Jiang, Bingjun, Han, Tianfu, Hou, Wensheng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321276/
https://www.ncbi.nlm.nih.gov/pubmed/30513774
http://dx.doi.org/10.3390/ijms19123835
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author Cai, Yupeng
Chen, Li
Sun, Shi
Wu, Cunxiang
Yao, Weiwei
Jiang, Bingjun
Han, Tianfu
Hou, Wensheng
author_facet Cai, Yupeng
Chen, Li
Sun, Shi
Wu, Cunxiang
Yao, Weiwei
Jiang, Bingjun
Han, Tianfu
Hou, Wensheng
author_sort Cai, Yupeng
collection PubMed
description At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for complex genome editing, especially the deletion of large DNA fragments in soybean, has not been systematically explored. In this study, we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2a. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2a and 15.8% for target fragments ranging from 1069 to 1161 bp in GmFT5a. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 ‘transgene-free’ homozygous ft2a mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean.
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spelling pubmed-63212762019-01-07 CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean Cai, Yupeng Chen, Li Sun, Shi Wu, Cunxiang Yao, Weiwei Jiang, Bingjun Han, Tianfu Hou, Wensheng Int J Mol Sci Article At present, the application of CRISPR/Cas9 in soybean (Glycine max (L.) Merr.) has been mainly focused on knocking out target genes, and most site-directed mutagenesis has occurred at single cleavage sites and resulted in short deletions and/or insertions. However, the use of multiple guide RNAs for complex genome editing, especially the deletion of large DNA fragments in soybean, has not been systematically explored. In this study, we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2a. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2a and 15.8% for target fragments ranging from 1069 to 1161 bp in GmFT5a. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 ‘transgene-free’ homozygous ft2a mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean. MDPI 2018-12-01 /pmc/articles/PMC6321276/ /pubmed/30513774 http://dx.doi.org/10.3390/ijms19123835 Text en © 2018 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
Cai, Yupeng
Chen, Li
Sun, Shi
Wu, Cunxiang
Yao, Weiwei
Jiang, Bingjun
Han, Tianfu
Hou, Wensheng
CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean
title CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean
title_full CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean
title_fullStr CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean
title_full_unstemmed CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean
title_short CRISPR/Cas9-Mediated Deletion of Large Genomic Fragments in Soybean
title_sort crispr/cas9-mediated deletion of large genomic fragments in soybean
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321276/
https://www.ncbi.nlm.nih.gov/pubmed/30513774
http://dx.doi.org/10.3390/ijms19123835
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