Cargando…
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...
Autores principales: | , , , , , , , |
---|---|
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 |
_version_ | 1783385403404845056 |
---|---|
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. |
format | Online Article Text |
id | pubmed-6321276 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT caiyupeng crisprcas9mediateddeletionoflargegenomicfragmentsinsoybean AT chenli crisprcas9mediateddeletionoflargegenomicfragmentsinsoybean AT sunshi crisprcas9mediateddeletionoflargegenomicfragmentsinsoybean AT wucunxiang crisprcas9mediateddeletionoflargegenomicfragmentsinsoybean AT yaoweiwei crisprcas9mediateddeletionoflargegenomicfragmentsinsoybean AT jiangbingjun crisprcas9mediateddeletionoflargegenomicfragmentsinsoybean AT hantianfu crisprcas9mediateddeletionoflargegenomicfragmentsinsoybean AT houwensheng crisprcas9mediateddeletionoflargegenomicfragmentsinsoybean |