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Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus
- 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.
- Subjects :
- CLCuD
QH301-705.5
Agrobacterium
Genetically modified crops
cotton
Catalysis
Article
virus inhibition
Inorganic Chemistry
CRISPR/Cas
CRISPR
DsRed
Biology (General)
Physical and Theoretical Chemistry
QD1-999
Molecular Biology
Spectroscopy
Solanaceae
Disease Resistance
Plant Diseases
Gossypium
biology
Cas9
N. benthamiana
transformation
Organic Chemistry
fungi
food and beverages
General Medicine
biology.organism_classification
Plants, Genetically Modified
Virology
Plant disease
Computer Science Applications
Chemistry
Transformation (genetics)
Cotton leaf curl virus
Begomovirus
Leaf curl
CRISPR-Cas Systems
Subjects
Details
- Language :
- English
- ISSN :
- 14220067
- Volume :
- 22
- Issue :
- 22
- Database :
- OpenAIRE
- Journal :
- International Journal of Molecular Sciences
- Accession number :
- edsair.doi.dedup.....680590f9012592911942d66840202724