Department of Plant Pathology, Kansas State University, Manhattan, KS, USA.
Methods Mol Biol. 2021;2170:1-18. doi: 10.1007/978-1-0716-0743-5_1.
Application of the CRISPR-Cas prokaryotic immune system for single-stranded RNA targeting will have significant impacts on RNA analysis and engineering. The class 2 Type VI CRISPR-Cas13 system is an RNA-guided RNA-nuclease system capable of binding and cleaving target single-stranded RNA substrates in a sequence-specific manner. In addition to RNA interference, the Cas13a system has application from manipulating RNA modifications, to editing RNA sequence, to use as a nucleic acid detection tool. This protocol uses the Cas13a ortholog from Leptotrichia buccalis for transient expression in plant cells providing antiviral defense. We cover all the necessary information for cloning the Cas13 protein, crRNA guide cassette, performing transient Agrobacterium-mediated expression of the necessary Cas13a components and target RNA-virus, visualization of virus infection, and molecular quantification of viral accumulation using quantitative PCR.
CRISPR-Cas 原核免疫系统在单链 RNA 靶向中的应用将对 RNA 分析和工程产生重大影响。II 类 VI 型 CRISPR-Cas13 系统是一种 RNA 指导的 RNA 核酸酶系统，能够以序列特异性方式结合并切割靶标单链 RNA 底物。除了 RNA 干扰外，Cas13a 系统还可用于 RNA 修饰操作、RNA 序列编辑，以及用作核酸检测工具。本方案使用 Leptotrichia buccalis 的 Cas13a 同源物在植物细胞中进行瞬时表达，以提供抗病毒防御。我们涵盖了克隆 Cas13 蛋白、crRNA 引导盒、通过瞬时农杆菌介导表达必要的 Cas13a 成分和靶标 RNA 病毒、病毒感染的可视化以及使用定量 PCR 进行病毒积累的分子定量所需的所有必要信息。
