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

Area of Science:

• Plant Biotechnology
• Genomics
• Agricultural Science

Background:

• Cotton production in Pakistan is declining due to factors including insect pests, cotton leaf curl disease (CLCuD), and abiotic stresses.
• Cotton leaf curl disease (CLCuD) is a significant threat to cotton yield in Pakistan.
• CRISPR/Cas9 genome editing offers a novel approach for developing plant immunity against viral diseases.

Purpose of the Study:

• To demonstrate the efficacy of multiplex CRISPR/Cas-mediated genome editing against cotton leaf curl viruses (CLCuVs).
• To assess the potential of this technology for developing CLCuD-resistant cotton varieties.

Main Methods:

• CRISPR/Cas9 system was employed for multiplex genome editing targeting three regions of the CLCuV genome.
• Guide RNAs (gRNAs) were designed, cloned into Cas9-expressing vectors, and confirmed via PCR, restriction analysis, and sequencing.
• Transient transformation was performed in *Nicotiana benthamiana* and cotton seedlings using *Agrobacterium*-mediated infiltration.

Main Results:

• Transient expression in *N. benthamiana* resulted in delayed CLCuD symptoms (3-5 days) and improved resistance.
• Viral titer was reduced by 20-40% in plants treated with Cas9-gRNA compared to controls.
• Similar resistance was observed in cotton seedlings, with five transgenic plants showing 60-70% resistance to CLCuV.

Conclusions:

• Multiplex CRISPR/Cas genome editing is a viable strategy for developing resistance against CLCuD.
• This technology holds significant potential for breeding CLCuD-resistant cotton varieties, contributing to sustainable cotton production.
• Further development of transgenic plants can lead to improved crop resilience and agricultural sustainability.