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:	Barkha Binyameen, Zulqurnain Khan, Sultan Habibullah Khan, Aftab Ahmad, Nayla Munawar, Muhammad Salman Mubarik, Hasan Riaz, Zulfiqar Ali, Asif Ali Khan, Alaa T. Qusmani, Kamel A. Abd-Elsalam, Sameer H. Qari
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	cotton CLCuD CRISPR/Cas <i>N. benthamiana</i> virus inhibition DsRed Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/cffe0899f0cd48508cca9cbde4faafac
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spelling	oai:doaj.org-article:cffe0899f0cd48508cca9cbde4faafac2021-11-25T17:57:40ZUsing Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus10.3390/ijms2222125431422-00671661-6596https://doaj.org/article/cffe0899f0cd48508cca9cbde4faafac2021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/22/12543https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067In 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 <i>N. benthamiana</i> 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 <i>N. benthamiana</i> 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 <i>N. benthamiana</i> 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.Barkha BinyameenZulqurnain KhanSultan Habibullah KhanAftab AhmadNayla MunawarMuhammad Salman MubarikHasan RiazZulfiqar AliAsif Ali KhanAlaa T. QusmaniKamel A. Abd-ElsalamSameer H. QariMDPI AGarticlecottonCLCuDCRISPR/Cas<i>N. benthamiana</i>virus inhibitionDsRedBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 12543, p 12543 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	cotton CLCuD CRISPR/Cas <i>N. benthamiana</i> virus inhibition DsRed Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	cotton CLCuD CRISPR/Cas <i>N. benthamiana</i> virus inhibition DsRed Biology (General) QH301-705.5 Chemistry QD1-999 Barkha Binyameen Zulqurnain Khan Sultan Habibullah Khan Aftab Ahmad Nayla Munawar Muhammad Salman Mubarik Hasan Riaz Zulfiqar Ali Asif Ali Khan Alaa T. Qusmani Kamel A. Abd-Elsalam Sameer H. Qari Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus
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 <i>N. benthamiana</i> 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 <i>N. benthamiana</i> 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 <i>N. benthamiana</i> 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	article
author	Barkha Binyameen Zulqurnain Khan Sultan Habibullah Khan Aftab Ahmad Nayla Munawar Muhammad Salman Mubarik Hasan Riaz Zulfiqar Ali Asif Ali Khan Alaa T. Qusmani Kamel A. Abd-Elsalam Sameer H. Qari
author_facet	Barkha Binyameen Zulqurnain Khan Sultan Habibullah Khan Aftab Ahmad Nayla Munawar Muhammad Salman Mubarik Hasan Riaz Zulfiqar Ali Asif Ali Khan Alaa T. Qusmani Kamel A. Abd-Elsalam Sameer H. Qari
author_sort	Barkha Binyameen
title	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_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_sort	using multiplexed crispr/cas9 for suppression of cotton leaf curl virus
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/cffe0899f0cd48508cca9cbde4faafac
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Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus

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