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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...

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Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
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.
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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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