Karimi Marzieh, Ghorbani Abozar, Niazi Ali, Rostami Mahsa, Tahmasebi Ahmad
Institute of Biotechnology, Shiraz University, Shiraz, Iran.
Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute (NSTRI), Karaj, Iran.
Sci Rep. 2025 Jul 14;15(1):25422. doi: 10.1038/s41598-025-11405-z.
The Tomato Brown Rugose Fruit Virus (ToBRFV) has recently emerged as a serious threat to global tomato production, underscoring the need for rapid and sensitive diagnostic tools. Here, we present an AI-driven CRISPR-Cas13a pipeline for designing crRNAs with high specificity to enable the detection of ToBRFV. A computational pipeline that retrieves viral sequences, aligns them in multiple sequence alignments, analyzes their conservation, and screens for off-targets-all coupled with machine learning to optimize crRNA sequences. Experimentally validated crRNAs were evaluated with a fluorescence-based Cas13a assay and showed better sensitivity than RT-PCR, RT-qPCR, and RT-LAMP. By the CRISPR-Cas13a system, ToBRFV was detected at 1:200 (1 ng/µL) dilutions, which performed superior to conventional methods. Integrating bioinformatics with experimental workflows, this pipeline provides a powerful framework for rapid diagnostics that can be deployed in the field, addressing significant challenges in plant virus surveillance and management.
番茄褐色皱纹果病毒（ToBRFV）最近已成为全球番茄生产的严重威胁，这凸显了对快速且灵敏的诊断工具的需求。在此，我们展示了一种由人工智能驱动的CRISPR-Cas13a流程，用于设计具有高特异性的crRNA，以实现对ToBRFV的检测。该计算流程可检索病毒序列、将它们进行多序列比对、分析其保守性并筛选脱靶情况，所有这些都与机器学习相结合以优化crRNA序列。经实验验证的crRNA通过基于荧光的Cas13a检测进行评估，结果显示其灵敏度优于逆转录聚合酶链反应（RT-PCR）、逆转录定量聚合酶链反应（RT-qPCR）和逆转录环介导等温扩增技术（RT-LAMP）。通过CRISPR-Cas13a系统，在1:200（1纳克/微升）稀释度下检测到了ToBRFV，其表现优于传统方法。该流程将生物信息学与实验工作流程相结合，为可在现场部署的快速诊断提供了一个强大框架，解决了植物病毒监测和管理中的重大挑战。
