Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA₃) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.

中文翻译：

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RNA靶向III型CRISPR复合物与NucC核酸内切酶效应物的特异性和敏感性

III 型 CRISPR 系统检测入侵的 RNA，导致酶促 Cas10 亚基的激活。Cas10 环化酶结构域产生环状寡腺苷酸 (cOA) 第二信使分子，激活多种效应核酸酶，降解核酸以提供免疫力。原噬菌体编码的弧菌III-B 型 (VmeCmr) 基因座没有特征，在 Cas10 中缺少 HD 核酸酶结构域，并编码一个 NucC DNA 核酸酶效应子，该效应子也被发现与基于环寡核苷酸的抗噬菌体信号系统 (CBASS) 相关. 在这里我们证明 VmeCmr 被目标 RNA 结合激活，产生环三腺苷酸 (cA ₃) 以刺激强大的 NucC 介导的 DNase 活性。探测 VmeCmr 的特异性，揭示了 RNA 双链体第 1 段和原型间隔区侧翼序列 (PFS) 中特定核苷酸位置的重要性。我们利用这个可编程系统证明了使用商业读板器检测 SARS-CoV-2 病毒 RNA 的高度特异性和灵敏性检测的潜力，检测限 (LoD) 为 2 fM，无需任何外在扩增步骤。灵敏度高度依赖于所使用的引导 RNA，表明目标 RNA 二级结构起着重要的作用，在体内也可能相关。