Cargando…

Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant

Upland cotton (Gossypium hirsutum), an allotetraploid, contains At- and Dt- subgenome and most genes have multiple homologous copies, which pose a huge challenge to investigate genes’ function due to the functional redundancy. Therefore, it is of great significance to establish effective techniques...

Descripción completa

Detalles Bibliográficos
Autores principales: Li, Bo, Fu, Chunyang, Zhou, Jiawei, Hui, Fengjiao, Wang, Qiongqiong, Wang, Fuqiu, Wang, Guanying, Xu, Zhongping, Che, Lianlian, Yuan, Daojun, Wang, Yanqin, Zhang, Xianlong, Jin, Shuangxia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9496795/
https://www.ncbi.nlm.nih.gov/pubmed/36139477
http://dx.doi.org/10.3390/cells11182902
_version_ 1784794357624209408
author Li, Bo
Fu, Chunyang
Zhou, Jiawei
Hui, Fengjiao
Wang, Qiongqiong
Wang, Fuqiu
Wang, Guanying
Xu, Zhongping
Che, Lianlian
Yuan, Daojun
Wang, Yanqin
Zhang, Xianlong
Jin, Shuangxia
author_facet Li, Bo
Fu, Chunyang
Zhou, Jiawei
Hui, Fengjiao
Wang, Qiongqiong
Wang, Fuqiu
Wang, Guanying
Xu, Zhongping
Che, Lianlian
Yuan, Daojun
Wang, Yanqin
Zhang, Xianlong
Jin, Shuangxia
author_sort Li, Bo
collection PubMed
description Upland cotton (Gossypium hirsutum), an allotetraploid, contains At- and Dt- subgenome and most genes have multiple homologous copies, which pose a huge challenge to investigate genes’ function due to the functional redundancy. Therefore, it is of great significance to establish effective techniques for the functional genomics in cotton. In this study, we tested two novel genome editing vectors and compared them with the CRISPR/Cas9 system (pRGEB32-GhU6.7) developed in our laboratory previously. In the first new vector, the sgRNA transcription unite was constructed into the replicon (LIR-Donor-SIR-Rep-LIR) of the bean yellow dwarf virus (BeYDV) and named as pBeYDV-Cas9-KO and in the second vector, the ubiquitin promoter that drives Cas9 protein was replaced with a constitutive CaMV 35S promoter and defined as pRGEB32-35S. The results from transgenic cotton calli/plants revealed that pBeYDV-Cas9-KO vector showed the highest editing efficiency of GhCLA1 in At and Dt subgenomes edited simultaneously up to 73.3% compared to the 44.6% of pRGEB32-GhU6.7 and 51.2% of pRGEB32-35S. The editing efficiency of GhCLA1 in At and Dt subgenome by pBeYDV-Cas9-KO was 85.7% and 97.2%, respectively, whereas the efficiency by pRGEB32-GhU6.7 and pRGEB32-35S vectors was 67.7%, 86.5%, 84%, and 87.2%, respectively. The editing profile of pBeYDV-Cas9-KO was mainly composed of fragment deletion, accounting for 84.0% and ranging 1–10 bp in length. The main editing sites are located at positions 11–17 upstream of PAM site. The off-target effects were not detected in all potential off-target sites. Taken together, the pBeYDV-Cas9-KO system has high editing efficiency and specificity with wide editing range than the traditional CRISPR/Cas9 system, which provides a powerful tool for cotton functional genomics research and molecular breeding.
format Online
Article
Text
id pubmed-9496795
institution National Center for Biotechnology Information
language English
publishDate 2022
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-94967952022-09-23 Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant Li, Bo Fu, Chunyang Zhou, Jiawei Hui, Fengjiao Wang, Qiongqiong Wang, Fuqiu Wang, Guanying Xu, Zhongping Che, Lianlian Yuan, Daojun Wang, Yanqin Zhang, Xianlong Jin, Shuangxia Cells Article Upland cotton (Gossypium hirsutum), an allotetraploid, contains At- and Dt- subgenome and most genes have multiple homologous copies, which pose a huge challenge to investigate genes’ function due to the functional redundancy. Therefore, it is of great significance to establish effective techniques for the functional genomics in cotton. In this study, we tested two novel genome editing vectors and compared them with the CRISPR/Cas9 system (pRGEB32-GhU6.7) developed in our laboratory previously. In the first new vector, the sgRNA transcription unite was constructed into the replicon (LIR-Donor-SIR-Rep-LIR) of the bean yellow dwarf virus (BeYDV) and named as pBeYDV-Cas9-KO and in the second vector, the ubiquitin promoter that drives Cas9 protein was replaced with a constitutive CaMV 35S promoter and defined as pRGEB32-35S. The results from transgenic cotton calli/plants revealed that pBeYDV-Cas9-KO vector showed the highest editing efficiency of GhCLA1 in At and Dt subgenomes edited simultaneously up to 73.3% compared to the 44.6% of pRGEB32-GhU6.7 and 51.2% of pRGEB32-35S. The editing efficiency of GhCLA1 in At and Dt subgenome by pBeYDV-Cas9-KO was 85.7% and 97.2%, respectively, whereas the efficiency by pRGEB32-GhU6.7 and pRGEB32-35S vectors was 67.7%, 86.5%, 84%, and 87.2%, respectively. The editing profile of pBeYDV-Cas9-KO was mainly composed of fragment deletion, accounting for 84.0% and ranging 1–10 bp in length. The main editing sites are located at positions 11–17 upstream of PAM site. The off-target effects were not detected in all potential off-target sites. Taken together, the pBeYDV-Cas9-KO system has high editing efficiency and specificity with wide editing range than the traditional CRISPR/Cas9 system, which provides a powerful tool for cotton functional genomics research and molecular breeding. MDPI 2022-09-16 /pmc/articles/PMC9496795/ /pubmed/36139477 http://dx.doi.org/10.3390/cells11182902 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Li, Bo
Fu, Chunyang
Zhou, Jiawei
Hui, Fengjiao
Wang, Qiongqiong
Wang, Fuqiu
Wang, Guanying
Xu, Zhongping
Che, Lianlian
Yuan, Daojun
Wang, Yanqin
Zhang, Xianlong
Jin, Shuangxia
Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant
title Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant
title_full Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant
title_fullStr Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant
title_full_unstemmed Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant
title_short Highly Efficient Genome Editing Using Geminivirus-Based CRISPR/Cas9 System in Cotton Plant
title_sort highly efficient genome editing using geminivirus-based crispr/cas9 system in cotton plant
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9496795/
https://www.ncbi.nlm.nih.gov/pubmed/36139477
http://dx.doi.org/10.3390/cells11182902
work_keys_str_mv AT libo highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant
AT fuchunyang highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant
AT zhoujiawei highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant
AT huifengjiao highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant
AT wangqiongqiong highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant
AT wangfuqiu highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant
AT wangguanying highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant
AT xuzhongping highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant
AT chelianlian highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant
AT yuandaojun highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant
AT wangyanqin highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant
AT zhangxianlong highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant
AT jinshuangxia highlyefficientgenomeeditingusinggeminivirusbasedcrisprcas9systemincottonplant