Institute of Plant Breeding and Biotechnology, MNS University of Agriculture Multan, Old Shujabad Road, Multan 60000, Pakistan.
Cotton Biotechnology Lab, Center for Advanced Studies in Agriculture and Food Security, University of Agriculture, Faisalabad 38040, Pakistan.
Int J Mol Sci. 2021 Nov 21;22(22):12543. doi: 10.3390/ijms222212543.
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 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 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 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.
近几十年来，由于昆虫、棉花卷曲病毒（CLCuD）和多种非生物胁迫等多种因素，巴基斯坦的棉花产量下降。CLCuD 是一种严重危害植物的疾病，严重限制了巴基斯坦的棉花产量。最近，通过 CRISPR/Cas9 的基因组编辑技术彻底改变了植物生物学，特别是为植物对病毒性疾病产生免疫提供了可能。在这里，我们通过 和棉花幼苗的瞬时转化，展示了针对 CLCuD 的多重 CRISPR/Cas 介导的基因组编辑。我们从 NCBI 获得了棉花卷曲病毒（CLCuV）的基因组序列，并使用 CRISPR MultiTargeter 设计了靶向病毒基因组三个区域的 gRNA。gRNA 被克隆到 pHSE401/pKSE401 中，该载体包含 Cas9，并通过菌落 PCR、限制性分析和测序进行了验证。确认的构建体被转移到农杆菌中，然后用于转化。在 中进行的 agroinfilteration 显示出延迟的症状（3-5 天），对 CLCuD 的抗性得到改善。此外，与对照植株（仅感染病毒）相比，共侵染 Cas9-gRNA 的感染植株中的病毒滴度也较低（20-40%）。在棉花幼苗中也得到了类似的结果。在 和棉花幼苗中的瞬时表达结果表明，多重 CRISPR/Cas 具有开发抗 CLCuD 的潜力。从三个实验中开发的 5 株转基因植物对 CLCuV 表现出抗性（60-70%），其中在评估和筛选过程中选择了两个最佳植株。该技术将有助于培育抗 CLCuD 的棉花品种，以实现可持续的棉花生产。
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