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Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus

Authors :
Barkha Binyameen
Zulfiqar Ali
Kamel A. Abd-Elsalam
Aftab Ahmad
Alaa T. Qusmani
Asif Ali Khan
Muhammad Salman Mubarik
Zulqurnain Khan
Nayla Munawar
Sultan Habibullah Khan
Sameer H. Qari
Hasan Riaz
Source :
International Journal of Molecular Sciences, International Journal of Molecular Sciences; Volume 22; Issue 22; Pages: 12543, International Journal of Molecular Sciences, Vol 22, Iss 12543, p 12543 (2021)
Publication Year :
2021
Publisher :
MDPI, 2021.

Abstract

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.

Details

Language :
English
ISSN :
14220067
Volume :
22
Issue :
22
Database :
OpenAIRE
Journal :
International Journal of Molecular Sciences
Accession number :
edsair.doi.dedup.....680590f9012592911942d66840202724