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Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus
In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome edit...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8618328/ https://www.ncbi.nlm.nih.gov/pubmed/34830426 http://dx.doi.org/10.3390/ijms222212543 |
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author | Binyameen, Barkha Khan, Zulqurnain Khan, Sultan Habibullah Ahmad, Aftab Munawar, Nayla Mubarik, Muhammad Salman Riaz, Hasan Ali, Zulfiqar Khan, Asif Ali Qusmani, Alaa T. Abd-Elsalam, Kamel A. Qari, Sameer H. |
author_facet | Binyameen, Barkha Khan, Zulqurnain Khan, Sultan Habibullah Ahmad, Aftab Munawar, Nayla Mubarik, Muhammad Salman Riaz, Hasan Ali, Zulfiqar Khan, Asif Ali Qusmani, Alaa T. Abd-Elsalam, Kamel A. Qari, Sameer H. |
author_sort | Binyameen, Barkha |
collection | PubMed |
description | In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3–5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20–40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60−70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production. |
format | Online Article Text |
id | pubmed-8618328 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-86183282021-11-27 Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus Binyameen, Barkha Khan, Zulqurnain Khan, Sultan Habibullah Ahmad, Aftab Munawar, Nayla Mubarik, Muhammad Salman Riaz, Hasan Ali, Zulfiqar Khan, Asif Ali Qusmani, Alaa T. Abd-Elsalam, Kamel A. Qari, Sameer H. Int J Mol Sci Article In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3–5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20–40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60−70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production. MDPI 2021-11-21 /pmc/articles/PMC8618328/ /pubmed/34830426 http://dx.doi.org/10.3390/ijms222212543 Text en © 2021 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 Binyameen, Barkha Khan, Zulqurnain Khan, Sultan Habibullah Ahmad, Aftab Munawar, Nayla Mubarik, Muhammad Salman Riaz, Hasan Ali, Zulfiqar Khan, Asif Ali Qusmani, Alaa T. Abd-Elsalam, Kamel A. Qari, Sameer H. Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus |
title | Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus |
title_full | Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus |
title_fullStr | Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus |
title_full_unstemmed | Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus |
title_short | Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus |
title_sort | using multiplexed crispr/cas9 for suppression of cotton leaf curl virus |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8618328/ https://www.ncbi.nlm.nih.gov/pubmed/34830426 http://dx.doi.org/10.3390/ijms222212543 |
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